Puro-DHFR quadrifunctional marker and its use in protein production

ABSTRACT

This invention relates to industrial production of proteins. More specifically, the invention relates to the res-DHFR surrogate marker, which corresponds to a fusion between DHFR and a protein conferring resistance to a toxic compound or conferring a metabolic advantage. The invention further relates to the use of res-DHFR for screening cells for high expression of a protein of interest. The invention is illustrated by the Puro-DHFR surrogate marker, which corresponds to a fusion between the puromycin N-acetyltransferase and dihydrofolate reductase (DHFR).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/601,553, filed Nov. 24, 2009, now U.S. Pat. No. 8,357,535, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2008/057109, filed Jun. 6, 2008, which claims the benefit ofU.S. Provisional Patent Application No. 60/934,078, filed Jun. 11, 2007,the disclosures of which are hereby incorporated by reference in theirentirety, including all figures, tables and amino acid or nucleic acidsequences.

FIELD OF THE INVENTION

This invention relates to industrial production of proteins. Morespecifically, the invention relates to the res-DHFR surrogate marker,which corresponds to a fusion between DHFR and a protein conferringresistance to a toxic compound or conferring a metabolic advantage. Theinvention further relates to the use of res-DHFR for screening cells forhigh expression of a protein of interest. The invention is illustratedby the Puro-DHFR surrogate marker, which corresponds to a fusion betweenthe puromycin N-acetyltransferase and dihydrofolate reductase (DHFR).

BACKGROUND

Introducing heterologous genes into animal host cells and screening forexpression of the added genes is a lengthy and complicated process.Typically a number of hurdles have to be overcome: (i) the constructionof large expression vectors; (ii) the transfection and selection ofclones with stable long-term expression, eventually in the absence ofselective pressure; and (iii) screening for high expression rates of theheterologous protein of interest.

1. Selection of Clones Expressing the Heterologous Gene

Selection of the clones having integrated the gene of interest isperformed using a selection marker conferring resistance to a selectivepressure. Most of the selection markers confer resistance to anantibiotic such as, e.g., neomycin, kanamycin, hygromycin, gentamycin,chloramphenicol, puromycin, zeocin or bleomycin.

When generating cell clones expressing a gene of interest fromexpression vectors, host cells are typically transfected with a plasmidDNA vector encoding both the protein of interest and the selectionmarker on the same vector. Quite often the capacity of a plasmid islimited and the selection marker has to be expressed from a secondplasmid, which is co-transfected with the plasmid comprising the gene ofinterest.

Stable transfection leads to random integration of the expression vectorin the genome of the host cell. Use of selective pressure, e.g. byadministrating an antibiotic to the media, will eliminate all cells thatdid not integrate the vector containing the selection marker providingresistance to the respective antibiotic or selective pressure. If thisselection marker is on the same vector as the gene of interest or, ifthis selection marker is on a second vector and vector comprising thegene of interest was co-integrated, the cells will express both theselection marker and the gene of interest. It is frequently observed,however, that the expression level of the gene of interest is highlyvariable depending on the site of integration.

Furthermore, when removing selective pressure, expression becomes quiteoften very unstable or even extinguished. Only a small number of initialtransfectants are thus providing high and stable long-term expressionand it is time-consuming to identify these clones in a large populationof candidates. Typically, high expressing candidates are isolated andthen cultivated in absence of selective pressure. Under these conditionsa large proportion of initially selected candidates are eliminated dueto their loss of gene of interest expression upon removal of selectivepressure. It would thus be advantageous to cultivate the candidates,following an initial period of selection for stable transfection, inabsence of selective pressure and only then screen for gene of interestexpression.

2. Screening for High Expressing Clones

Screening for high-expressing clones for a protein of interest is oftendone by methods directly revealing the presence of high amounts of theprotein. Typically immunologic methods, such as ELISA orimmunohistochemical staining, are applied to detect the product eitherintracellularly or in cell culture supernatants. These methods aretedious, expensive, time-consuming, and often not amenable to highthroughput screenings (HTS). In addition, an antibody reactive to theexpressed protein must be available.

Attempts to quantify the protein amounts by Fluorescence-Activated CellSorting (FACS) have also been made, but only with a limited success,especially in the case of secreted proteins (Borth et al., 2000).

One approach for the screening of high expression rates of the proteinof interest would be the use of an easily measurable surrogate marker,expressed from the same vector as the gene of interest (Chesnut et al.,1996). The idea underlying the use of a measurable surrogate marker isthat there is a correlation between the expression of the gene ofinterest and the surrogate marker gene due to the physical link of thetwo genes on the same vector.

Numerous easily measurable markers are available in the art. Theyusually correspond to enzymes, which act on a chromogenic or luminogenicsubstrate such as, e.g., the β-glucuronidase, the chloramphenicolacetyltransferase, the nopaline synthase, the β-galactosidase, secretedalkaline phosphatase (SEAP) and the DHFR. The green fluorescent protein(GFP) may also be used as a measurable marker in FACS. The activity ofall these proteins can be measured by standard assays that may be usedin HTS.

The drawback of this approach is the use of yet another expressioncassette for the surrogate marker gene. This renders the expressionvector rather bulky, hosting expression units comprising a promoter, acDNA and polyadenylation signals for at least three proteins (i.e., thegene of interest, the selection marker and the surrogate marker). Formulti-chain proteins the situation becomes even more complex.Alternatively, individual plasmid vectors expressing the three genes,which encode the protein of interest, the selection marker and thesurrogate marker respectively, could be co-transfected. However, it islikely that the vectors would be either integrated at different loci, orexhibit varying and uncorrelated expression.

A promising approach for overcoming the above limitations consists inthe use of a chimeric marker that combines the functional properties ofa selection marker and of a measurable marker.

Such bifunctional markers have been described by, e.g., Bennett et al.(1998), Imhof and Chatellard (2006) and Dupraz and Kobr (2007). Bennettet al. (1998) disclose the GFP-Zeo^(R) marker, which confers resistanceto Zeocin antibiotic, which corresponds to a fusion protein between theGreen Fluorescent Protein (GFP) and a protein conferring resistance tozeocin. Imhof and Chatellard (2006) disclose the Lupac marker, whichcorresponds to a fusion between the firefly luciferase protein and aprotein conferring resistance to puromycin. Dupraz and Kobr (2007)discloses the PuroLT marker, which corresponds to a fusion proteinbetween the synthetic peptide described by Griffin et al. (1998) and aprotein conferring resistance to puromycin.

Miller et al. (2005), in an article showing that fluorescent TMP is analternative to fluorescent MTX, discloses a fusion protein between aprotein conferring resistance to puromycin and a DHFR of bacterialorigin. DHFR is used as measurable marker that can be detected bybinding to fluorescent MTX or to fluorescent TMP. This article envisionsthe use of the fusion protein in the field of siRNA gene silencing.

Hence, all markers available for the selection of clones expressing highlevels of a recombinant protein correspond to bifunctional markers,which confer resistance to a single toxic compound.

In addition to the bifunctional marker, the vectors used for generatinghigh producer clones usually comprise an amplifiable gene that leads toan increase in copy number when under selective pressure. The copynumber of a gene of interest positioned adjacent to the amplifiable genewill also increase, thus leading to the establishment of clonesexpressing high levels of the protein of interest (Kaufman et al., 1985;Kaufman et al., 1986; Kim et al., 2001; Omasa, 2002). Commonly usedamplifiable genes include e.g. dihydrofolate reductase (DHFR), Glutaminesynthetase (GS), multiple drug resistance gene (MDR), ornithinedecarboxylase (ODC), adenosine deaminase (ADA) andN-(phosphonacetyl)-L-aspartate resistance (CAD).

The finding of a novel and powerful chimeric surrogate marker,conferring resistance to more than one toxic compound and also allowinggene amplification, would be extremely useful in the field of industrialproduction of therapeutic proteins.

SUMMARY OF THE INVENTION

The present invention stems from the construction and characterizationof a novel quadrifunctional marker, Puro-DHFR. Puro-DHFR corresponds toa fusion protein between DHFR and a protein conferring resistance topuromycin, the puromycin N-acetyl transferase (pac). It has beendemonstrated that Puro-DHFR combines the functional properties of bothpac and DHFR. More specifically, Puro-DHFR is a quadrifunctional markerallowing to (i) select cells for resistance to puromycin; (ii) selectcells for resistance to DHFR; (iii) carry out gene amplification; and(iv) sort cells through fluorescence intensity. Puro-DHFR's usefulnessfor the isolation of high-expressing clones for a therapeutic proteinhas further been demonstrated.

Therefore, a first aspect of the invention relates to a method ofscreening cells for expression of a protein of interest, said methodcomprising the step of:

-   -   a) transfecting cells by an expression vector encoding (i) a        res-DHFR chimeric protein comprising a functional fragment of        dihydrofolate reductase (DHFR) fused to a fragment conferring        resistance to a toxic compound or conferring a metabolic        advantage; and (ii) a protein of interest;    -   b) selecting cells being resistant to said toxic compound or        gaining said metabolic advantage; and    -   c) assaying the fluorescence of the cells selected in step (ii)        with a fluorescent compound binding to DHFR,        wherein said protein conferring resistance to a toxic compound        or conferring a metabolic advantage is not DHFR.

A second aspect of the invention relates to a method of obtaining a cellline expressing a protein of interest, said method comprising the stepof:

-   -   a) screening cells according to a method of the invention; and    -   b) establishing a cell line from said cells.

A third aspect of the invention relates to a method of producing aprotein of interest, said method comprising the step of:

-   -   a) culturing a cell line obtained according to the above method        under conditions which permit expression of said protein of        interest; and    -   b) collecting said protein of interest.

A fourth aspect of the invention relates to a res-DHFR polypeptidecomprising a functional fragment of dihydrofolate reductase (DHFR) fusedto a fragment conferring resistance to a toxic compound or conferring ametabolic advantage, wherein said protein conferring resistance to atoxic compound or conferring a metabolic advantage is not DHFR.

A fifth aspect of the invention relates to a nucleic acid encoding ares-DHFR polypeptide in accordance with the invention.

A sixth aspect of the invention relates to a res-DHFR vector comprisinga nucleic acid in accordance with the invention.

A seventh aspect of the invention relates to a res-DHFR cell comprisinga nucleic acid in accordance with the invention.

Further aspects of the invention relate to the use of the res-DHFR cellof the invention for producing a protein of interest, to the use of ares-DHFR polypeptide for screening cells for expression of a protein ofinterest, to the use of a res-DHFR nucleic acid for screening cells forexpression of a protein of interest, and to the use of a res-DHFR vectorfor screening cells for expression of a protein of interest.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmids & reporter vectors used in the Examples.FIG. 1A.: pSV40-DHFR-1474; FIG. 1B.: pCMV(IE1)SEAP-IRES-Puro-279; FIG.1C.: pCMV(IE1)SEAP-IRES-Puro/DHFR-325. 1: gene conferring resistance toampicillin; 2: f1 origin of replication; 3: synthetic polyadenylationsignal; 4: SV40 promoter; 5: mCMV(IE1) promoter; 6: gene conferringresistance to DHFR; 7: SV40 polyadenylation signal; 8: SEAP gene; 9:poliovirus IRES; 10: gene conferring resistance to puromycin (puromycinN-acetyltransferase); 11: Puro-DHFR marker. All vectors further containthe ColE1-derived plasmid origin of replication.

FIG. 2 is a scheme representing the experiment for selecting clonestransfected with the plasmids & reporter vectors.

FIG. 3 compares the viability of DXB11-F10 CHO cells transfected withthe pCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector with the viability ofuntransfected DXB11-F10 CHO cells (control) during the selectionprocess. The experiment was performed in the absence of HT and in theabsence of puromycin (-HT), in the presence of HT and in the presence ofpuromycin at 10 mg/L (Puromycin), and in the absence of HT and in thepresence of puromycin (-HT/Puromycin).

FIG. 4 shows the productivity of alkaline phosphatase (SEAP) in batchcultures of DXB11-F10 CHO cells transfected with thepCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector. The experiment was performed inthe absence of HT and in the absence of puromycin (-HT), in the presenceof HT and in the presence of puromycin at 10 mg/L (Puromycin), in theabsence of HT and in the presence of puromycin (-HT/Puromycin), and inthe absence of HT and in the presence of MTX (50 nM) (-HT/MTX).

FIG. 5 shows the fluorescence of DXB11-F10 CHO cells transfected withthe pCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector grown in different selectionmedia and stained with fluorescent methotrexate (F-MTX).

FIGS. 6A-6C compare CHO-S cells transfected with thepCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector that encodes the puro-DHFRmarker (p325) with CHO-S cells transfected with thepCMV(IE1)SEAP-IRES-Puro-279 vector that encodes puromycin N-acetyltransferase (p279). FIG. 6A: Productivity of alkaline phosphatase (SEAP)FIG. 6B: Gene copy number of the marker (puro-DHFR or puromycin N-acetyltransferase) FIG. 6C: Relative SEAP mRNA expression level.

FIG. 7 shows the fluorescence of CHO-S cells transfected either with thepCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector (p325) or with thepCMV(IE1)SEAP-IRES-Puro-279 vector (p279) grown in different selectionmedia. “Phase contrast” corresponds to the optical technique used togenerate images of biological samples based on differences of therefractive index of the specimen in white light.

FIG. 8 shows the changes in Puro-DHFR copy number in clones of CHO-Scells stably transfected with pmCMV(IE1)—SEAP-IRES-Puro-DHFR-325 aftercultivation in selective medium containing either puromycin or puromycinplus 100 nM MTX.

BRIEF DESCRIPTION OF THE SEQUENCES OF THE INVENTION

SEQ ID Nos. 1 and 2 respectively correspond to the nucleic acid and tothe polypeptide sequences of a Puro-DHFR marker in accordance with theinvention.

SEQ ID Nos. 3 and 4 respectively correspond to the nucleic acid and tothe polypeptide sequences of Streptomyces alboniger puromycin N-acetyltransferase (pac).

SEQ ID Nos. 5 and 6 respectively correspond to the nucleic acid and tothe polypeptide sequences of murine DHFR.

SEQ ID Nos. 7 to 10 correspond to primers used when constructing thePuro-DHFR marker in accordance with the invention (Example 1).

SEQ ID Nos. 11 to 16 correspond to oligonucleotides used when detectingthe gene copy numbers by QPCR (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention stems from the construction and characterizationof a novel quadrifunctional chimeric marker referred to as res-DHFR. Theinvention more specifically discloses a res-DHFR polypeptide referred toas Puro-DHFR, which corresponds to a fusion protein between DHFR and aprotein conferring resistance to puromycin, the puromycin N-acetyltransferase (pac).

It has been demonstrated that Puro-DHFR is a quadrifunctional markerthat combines the functional properties of DHFR and of pac (Example 2).Accordingly, the Puro-DHFR marker can be used:

-   -   as a selectable marker in combination with the puromycin toxic        compound;    -   as a selectable marker in combination with the MTX toxic        compound;    -   as an amplifiable gene; and    -   as an easily measurable surrogate that can be detected both by        microscope and by FACS.

Puro-DHFR's usefulness for the isolation of high-expressing clones for aprotein of interest has further been demonstrated. In Example 3, avector comprising Puro-DHFR and a gene of interest, expressed from thesame promoter and separated by an IRES, has been constructed. It hasbeen shown that there is a very good positive correlation betweenPuro-DHFR expression levels and expression levels of the gene ofinterest.

Accordingly, the present invention provides powerful markers that can beused to provide selectivity in stable transfection, to induce geneamplification of the gene of interest, and which acts as a surrogatemarker for screening candidate clones for high expression of a gene ofinterest. Using res-DHFR, linked to a protein of interest in abicistronic configuration, allows keeping the same chance for selectinghigh-expressing clones as when the expression level of the gene ofinterest is measured directly. Moreover, using res-DHFR allows reducingtime, cost and resources since (i) standardized product-independent andsimple analysis is performed; and (ii) high expressors can be selectedusing a FACS.

1. Polypeptides of the Invention

The polypeptide according to the invention is a chimeric proteincomprising a functional fragment of a dihydrofolate reductase (DHFR)fused to a fragment conferring either resistance to a toxic compound ora metabolic advantage, wherein said fragment conferring resistance to atoxic compound or a metabolic advantage is not DHFR or a fragmentthereof. Such a chimeric protein will further be referred to as“polypeptide in accordance with the invention” or “res-DHFR” within thisspecification.

The fragment conferring resistance to a toxic compound may be selectedfrom the group consisting of a puromycin N-acetyltransferase (used incombination with the toxic compound puromycin), a neomycinphosphotransferase type II (used in combination with the toxic compoundneomycin), a kanamycin phosphotransferase type II (used in combinationwith the toxic compound kanamycin), a neomycin-kanamycinphosphotransferase type II (used in combination with the toxic compoundsneomycin and/or kanamycin), a hygromycin phosphotransferase (used incombination with the toxic compound hygromycin), a gentamycinacetyltransferase (used in combination with the toxic compoundgentamycin), a chloramphenicol acetyltransferase (used in combinationwith the toxic compound chloramphenicol), a zeocin resistance protein(used in combination with the toxic compound zeocin) and a bleomycinresistance protein (used in combination with the toxic compoundbleomycin).

In the frame of the present invention “a fragment conferring a metabolicadvantage” means that said fragment confers to a cell the ability togrow in the absence of a compound. For example, the glutamine synthetase(GS) protein confers to CHO cells the ability to grow in the absence ofglutamine. Thus the fragment conferring a metabolic advantage may e.g.correspond to glutamine synthetase (GS) or a functional fragmentthereof.

The term “functional fragment of DHFR” refers to a fragment of apolypeptide that is a member of the dihydrofolate reductase family (EC1.5.1.3), and that catalyzes the following enzymatic reaction:5,6,7,8-tetrahydrofolate+NADP⁺=7,8-dihydrofolate+NADPH

As used herein, the term “dihydrofolate reductase activity” refers tothe catalysis of the above reaction. This activity may be measured,e.g., by determining the ability to confer resistance to the toxiccompound methotrexate (MTX) to a cell as described in Example 1.2, or bydetermining the ability to increase the gene copy number in the presenceof MTX as described in Example 1.4. Alternatively, the DHFR activity canbe demonstrated by the ability of a DHFR-negative cell transfected withPuro-DHFR to grow in a medium devoid of thymidine and/or hypoxanthine.

In a preferred embodiment, the functional fragment of DHFR is derivedfrom mouse, and is a functional fragment of the sequence of SEQ ID NO:6. This fragment may comprise at least 50, 75, 100, 125, 150, 175 or 187amino acids of SEQ ID NO: 6. Most preferably, said functional fragmentof DHFR comprises amino acids 200 to 385 of SEQ ID NO: 2.

In a preferred embodiment of the invention, the res-DHFR polypeptide ofthe invention comprises a fragment of DHFR fused to a fragment of apuromycin N-acetyl transferase (pac), wherein said Puro-DHFR polypeptideexhibits (i) dihydrofolate reductase activity; and (ii) puromycinN-acetyl transferase activity. As further used herein, the term “aPuro-DHFR polypeptide” or “Puro-DHFR” refers to such a polypeptide.

As used herein, a polypeptide exhibits “puromycin N-acetyl transferaseactivity” when said polypeptide is capable of conferring resistance topuromycin to a cell. The puromycin N-acetyl transferase activity can forexample be measured as described in Example 1.2.

The fragment of a puromycin N-acetyl transferase may be derived from aStreptomyces species such as, e.g., Streptomyces alboniger orStreptomyces coelicolor. Preferably, the Puro-DHFR polypeptide comprisesa fragment of a Streptomyces alboniger pac. As used herein, the term“Streptomyces alboniger pac” refers to a polypeptide of SEQ ID NO: 4 orto an allelic variant, a splice variant or a mutein thereof. Morepreferably, the pac fragment comprises amino acids 1-199 of SEQ ID NO:2. Alternatively, said fragment of a Streptomyces alboniger pac cancomprise at least 50, 75, 100, 125, 150 or 175 amino acids of SEQ ID NO:4 as long as it retains puromycin N-acetyl transferase activity.

In a Puro-DHFR polypeptide, the DHFR fragment may be fused to the 3′terminus of the pac fragment, or the pac fragment may be fused to the 3′terminus of the DHFR fragment. Preferably, the DHFR fragment is fused tothe 3′ terminus of the pac fragment.

In a most preferred embodiment, the Puro-DHFR polypeptide comprises orconsists of SEQ ID NO: 2.

In another most preferred embodiment, the Puro-DHFR polypeptidecomprises or consists of an amino acid sequence at least 50% identical,more preferably at least 60% identical, and still more preferably atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 2.

As used herein, the term “mutein” refers to an analog of a naturallyoccurring polypeptide, in which one or more of the amino acid residuesof a naturally occurring polypeptide are replaced by different aminoacid residues, or are deleted, or one or more amino acid residues areadded to the naturally occurring sequence of the polypeptide, withoutlowering considerably the activity of the resulting products as comparedwith the naturally occurring polypeptide. These muteins are prepared byknown synthesis and/or by site-directed mutagenesis techniques, or anyother known technique suitable therefore. Muteins of Streptomycesalboniger pac or of murine DHFR that can be used in accordance with thepresent invention, or nucleic acids encoding the muteins, including afinite set of substantially corresponding sequences as substitutionpeptides or polynucleotides which can be routinely obtained by one ofordinary skill in the art, without undue experimentation, based on theteachings and guidance presented herein.

Muteins of Streptomyces alboniger pac or of murine DHFR in accordancewith the present invention include proteins encoded by a nucleic acid,such as DNA or RNA, which hybridizes to DNA or RNA, which encodes pac orDHFR, in accordance with the present invention, under moderately orhighly stringent conditions. The term “stringent conditions” refers tohybridization and subsequent washing conditions, which those of ordinaryskill in the art conventionally refer to as “stringent”. See Ausubel etal., Current Protocols in Molecular Biology, supra, Interscience, N.Y.,§6.3 and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J. C.,Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washingconditions 12-20° C. below the calculated Tm of the hybrid under studyin, e.g., 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinaryskill in this art understand that stringency conditions also depend onthe length of the DNA sequences, oligonucleotide probes (such as 10-40bases) or mixed oligonucleotide probes. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC.

Muteins of Streptomyces alboniger pac or of murine DHFR includepolypeptides having an amino acid sequence at least 50% identical, morepreferably at least 60% identical, and still more preferably at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to thenaturally occurring polypeptide.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

For sequences where there is not an exact correspondence, a “% identity”may be determined. In general, the two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orvery similar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. Thus for instance, programs available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux et al.,1984), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identityand the % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of Smith and Waterman (1981) and finds thebest single region of similarity between two sequences. Other programsfor determining identity and/or similarity between sequences are alsoknown in the art, for instance the BLAST family of programs (Altschul etal., 1990), accessible through the home page of the NCBI at world wideweb site ncbi.nlm.nih.gov) and FASTA (Pearson and Lipman, 1988; Pearson,1990). It is highly preferred that the % identity between two sequencesis determined using the KERR algorithm (Dufresne et al., 2002), forexample by using a bioinformatic tool such as e.g. GenePAST.

Preferably, the muteins of the present invention exhibit substantiallythe same biological activity as the naturally occurring polypeptide towhich it corresponds.

2. Nucleic Acids, and Vectors and Host Cells Comprising Them

Another aspect of the present invention relates to a nucleic acid thatencodes a res-DHFR polypeptide according to the invention.

Preferably, the nucleic acid according to the invention encodes aPuro-DHFR polypeptide. As further used in this specification, the term“Puro-DHFR nucleic acid” refers to such a nucleic acid.

In a preferred embodiment, the Puro-DHFR nucleic acid comprises orconsists of SEQ ID NO: 1.

Any procedures known in the art can be used to obtain Puro-DHFR nucleicacids of the present invention. Puro-DHFR nucleic acids can for examplebe obtained as described in Example 1.

A further aspect of the present invention relates to a vector comprisinga nucleic acid in accordance with the invention. A vector comprising ares-DHFR nucleic acid is referred to as a “res-DHFR vector”. A vectorcomprising a Puro-DHFR nucleic acid is referred to as a “Puro-DHFRvector” within the present specification. Preferably, the vector of theinvention is an expression vector. The term “vector of the invention”encompasses the term “Puro-DHFR vector”.

The term “vector” is used herein to designate either a circular or alinear DNA or RNA compound, which is either double-stranded orsingle-stranded, and which comprise at least one polynucleotide of thepresent invention to be transferred in a cell host or in a unicellularor multicellular host organism. An “expression vector” comprisesappropriate signals in the vectors, said signals including variousregulatory elements, such as enhancers/promoters from viral, bacterial,plant, mammalian, and other eucaryotic sources that drive expression ofthe inserted polynucleotide in host cells.

In a most preferred embodiment, the vector of the invention furthercomprises a nucleic acid encoding a protein of interest. As shown inexample 3, such vectors are particularly useful for screening cells forhigh expression of said protein of interest.

In accordance with the present invention, the protein of interest may beany polypeptide for which production is desired. The protein of interestmay find use in the field of pharmaceutics, agribusiness or furniturefor research laboratories. Preferred proteins of interests find use inthe field of pharmaceutics.

For example, the protein of interest may be, e.g., a naturally secretedprotein, a normally cytoplasmic protein, a normally transmembraneprotein, or a human or a humanized antibody. When the protein ofinterest is a normally cytoplasmic or a normally transmembrane protein,the protein has preferably been engineered in order to become soluble.The polypeptide of interest may be of any origin. Preferred polypeptidesof interest are of human origin.

In preferred embodiments, the protein of interest is selected from thegroup consisting of chorionic gonadotropin, follicle-stimulatinghormone, lutropin-choriogonadotropic hormone, thyroid stimulatinghormone, human growth hormone, interferons (e.g., interferon beta-1a,interferon beta-1b), interferon receptors (e.g., interferon gammareceptor), TNF receptors p55 and p75, interleukins (e.g., interleukin-2,interleukin-11), interleukin binding proteins (e.g., interleukin-18binding protein), anti-CD11a antibodies, erythropoietin, granulocytecolony stimulating factor, granulocyte-macrophage colony-stimulatingfactor, pituitary peptide hormones, menopausal gonadotropin,insulin-like growth factors (e.g., somatomedin-C), keratinocyte growthfactor, glial cell line-derived neurotrophic factor, thrombomodulin,basic fibroblast growth factor, insulin, Factor VIII, somatropin, bonemorphogenetic protein-2, platelet-derived growth factor, hirudin,epoietin, recombinant LFA-3/IgG1 fusion protein, glucocerebrosidase,monoclonal antibodies, and muteins, fragments, soluble forms, functionalderivatives, fusion proteins thereof.

Preferably, said monoclonal antibody is directed against a proteinselected from the group consisting of CD3 (e.g. OKT3, NI-0401), CD11a(e.g. efalizumab), CD4 (e.g. zanolimumab, TNX-355), CD20 (e.g.ibritumomab tiuxetan, rituximab, tositumomab, ocrelizumab, ofatumumab,IMMU-106, TRU-015, AME-133, GA-101), CD 23 (e.g. lumiliximab), CD22(e.g. epratuzumab), CD25 (e.g. basiliximab, daclizumab), the epidermalgrowth factor receptor (EGFR) (e.g. panitumumab, cetuximab, zalutumumab,MDX-214), CD30 (e.g MDX-060), the cell surface glycoprotein CD52 (e.g.alemtuzumab), CD80 (e.g. galiximab), the platelet GPIIb/IIIa receptor(e.g. abciximab), TNF alpha (e.g. infliximab, adalimumab, golimumab),the interleukin-6 receptor (e.g. tocilizumab,), carcinoembryonic antigen(CEA) (e.g. 99 mTc-besilesomab), alpha-4/beta-1 integrin (VLA4) (e.g.natalizumab), alpha-5/beta-1 integrin (VLA5) (e.g. volociximab), VEGF(e.g. bevacizumab, ranibizumab), immunoglobulin E (IgE) (e.g.omalizumab), HER-2/neu (e.g. trastuzumab), the prostate specificmembrane antigen (PSMA) (e.g. 111In-capromab pendetide, MDX-070), CD33(e.g. gemtuzumab ozogamicin), GM-CSF (e.g. KB002, MT203), GM-CSFreceptor (e.g. CAM-3001), EpCAM (e.g. adecatumumab), IFN-gamma (e.g.NI-0501), IFN-alpha (e.g. MEDI-545/MDX-1103), RANKL (e.g. denosumab),hepatocyte growth factor (e.g. AMG 102), IL-15 (e.g. AMG 714), TRAIL(e.g. AMG 655), insulin-like growth factor receptor (e.g. AMG 479,R1507), IL-4 and IL13 (e.g. AMG 317), BAFF/BLyS receptor 3 (BR3) (e.g.CB1), CTLA-4 (e.g. ipilimumab).

In a preferred embodiment, the vector of the invention is a nucleic acidencoding a protein of interest and comprising at least two promoters,one driving the expression of the polypeptide of the invention, and theother one driving the expression of the protein of interest. Such avector may further comprise enhancer regions, and/or expressionpromoting sequences such as insulators, boundary elements, LCRs (e.g.described by Blackwood and Kadonaga (1998) or matrix/scaffold attachmentregions (e.g. described by Li et al. (1999).

Alternatively, the vector of the invention comprises a promoter thatdrives both the expression of the gene of interest and the expression ofthe polypeptide of the invention. In this embodiment the ORF of thepolypeptide of the invention is separated from the ORF of the protein ofinterest by the presence of sequences such as e.g. an internal ribosomalentry sites (IRES) or a 2A sequence (de Felipe et al., 2006). When a 2Asequence is used, it is preferred that the Puro-DHFR corresponds to thefirst ORF (i.e. after the promoter) and that the protein of interestcorresponds to the second ORF (i.e. after the 2A sequence). The IRES maybe derived from, e.g., a virus or a cellular gene. This embodiment isexemplified by the pCMV(IE1)SEAP-IRES-Puro/DHFR-325 vector shown on FIG.1C, wherein the expression of SEAP gene (8) and of the Puro-DHFR marker(11) is driven by the mCMV(IE1) promoter (5), and wherein the ORFs areseparated by an IRES (9).

The term “promoter” as used herein refers to a region of DNA thatfunctions to control the transcription of one or more DNA sequences, andthat is structurally identified by the presence of a binding site forDNA-dependent RNA-polymerase and of other DNA sequences, which interactto regulate promoter function. A functional expression promotingfragment of a promoter is a shortened or truncated promoter sequenceretaining the activity as a promoter. Promoter activity may be measuredin any of the assays known in the art, e.g. in a reporter assay usingDHFR as reporter gene (Wood et al., 1984; SELIGER and McELROY, 1960; deWet et al., 1985), or commercially available from Promega®. An “enhancerregion” refers to a region of DNA that functions to increase thetranscription of one or more genes. More specifically, the term“enhancer”, as used herein, is a DNA regulatory element that enhances,augments, improves, or ameliorates expression of a gene irrespective ofits location and orientation vis-à-vis the gene to be expressed, and maybe enhancing, augmenting, improving, or ameliorating expression of morethan one promoter.

In a preferred embodiment, the vector of the invention comprises atleast one promoter of the murine CMV immediate early region. Thepromoter may for example be the promoter of the mCMV IE1 gene (the “IE1promoter”), which is known from, e.g., WO 87/03905. The promoter mayalso be the promoter of the mCMV IE2 gene (the “IE2 promoter”), the mCMVIE2 gene itself being known from, e.g., Messerle et al. (1991). The IE2promoter and the IE2 enhancer regions are described in details inPCT/EP2004/050280. Preferably, the vector of the invention comprises atleast two promoters of the murine CMV immediate early region. Morepreferably, the two promoters are the IE1 and the IE2 promoters.

In a preferred embodiment, the vector of the invention comprises atleast two promoters of the murine CMV immediate early region, whereinone of them drives the expression of a polypeptide of the invention, andthe other one drives the expression of a protein of interest.

In another preferred embodiment, the promoters of the murine CMVimmediate early region drive the expression of genes encoding a proteinof interest, and the Puro-DHFR polypeptide is expressed from anadditional expression cassette inserted in the vector backbone. The IE1and IE2 promoters may drive the expression either of two identicalcopies of the gene encoding the protein of interest, or of two subunitsof a multimeric protein of interest such as antibodies or peptidehormones.

Another aspect of the invention relates to a cell transfected with ares-DHFR nucleic acid of the invention and/or with a res-DHFR vector ofthe invention. Preferably, said cell is a Puro-DHFR cell transfectedwith a Puro-DHFR nucleic acid and/or a Puro-DHFR vector. Many cells aresuitable in accordance with the present invention, such as primary orestablished cell lines from a wide variety of eukaryotes including plantand animal cells. Preferably, said cell is a eukaryotic cell. Morepreferably, said cell is a mammalian cell. Most preferably, said cell isa CHO cell, a human cell, a mouse cell or a hybridoma.

For example, suitable cells include NIH-3T3 cells, COS cells, MRC-5cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO-Hep BSurface Antigen cells, HEK 293 cells, rHEK 293 cells, rC127-Hep BSurface Antigen cells, CV1 cells, mouse L cells, HT1080 cells, LM cells,Yl cells, NS0 and SP2/0 mouse hybridoma cells and the like, RPMI-8226cells, Vero cells, WI-38 cells, MRC-5 cells, Normal Human fibroblastcells, Human stroma cells, Human hepatocyte cells, human osteosarcomacells, Namalwa cells, human neuronal cells, human retinoblast cells,PER.C6 cells and other immortalized and/or transformed mammalian cells.

3. Methods of Using the Above Polypeptides and Nucleic Acids

Another aspect relates to the use of a cell comprising a res-DHFRnucleic acid according to the invention for producing a protein ofinterest. Preferably, said cell comprises a Puro-DHFR vector.

As discussed in Example 3, using a Puro-DHFR polypeptide as a selectionand surrogate marker provides numerous advantages for screening cellsfor high expression of a protein of interest. Specifically, since theexpression of the Puro-DHFR polypeptide is highly correlated with theexpression of the protein of interest, it is advantageous to perform aprimary screen for high Puro-DHFR expression, e.g. by FACS. Theexpression of the protein of interest is assayed in a secondary screen,which is only performed with the best producers isolated further to theprimary screen for high Puro-DHFR expression.

Accordingly, another aspect of the invention relates to the use of apolypeptide according to the invention, of a nucleic acid according tothe invention or of a vector according to the invention for screeningcells for expression or for high expression of a protein of interest.The cells are first screened for high expression of the polypeptideaccording to the invention (e.g. Puro-DHFR), and expression of thepolypeptide according to the invention is then correlated to that of aprotein of interest by inference. This allows to rapidly eliminate 80 to95% of the tested cells based on low expression levels of thepolypeptide according to the invention, and to retain the remaining5-20% for analysis of expression of the gene of interest in a secondstep.

In the context of the uses and methods of the present invention, theterm “high expression” refers to an expression level in a cell that isscreened that is higher than in other cells that are screened. “Highexpression” of a protein is a relative value. For example, finalexpression levels of recombinant proteins that are commercially produceddepend on the protein, annual quantities required and therapeutic dose.During a screening, the expression level of a protein of interest islower than the final expression level.

A further aspect relates to a method of screening cells for expressionor high expression of a protein of interest, said method comprising thestep of:

-   -   a) transfecting cells by an expression vector encoding res-DHFR;    -   b) selecting cells being resistant to said toxic compound; and    -   c) assaying the fluorescence of the cells selected in step (b)        with a fluorescent compound binding to DHFR.

Preferably, this method of screening cells for expression or highexpression of a protein of interest further comprising the step ofamplifying said recombinant protein of interest before performing step(c). Such an amplifying step is preferably performed by growing thecells in the presence of methotrexate (MTX). The concentration ofmethotrexate will vary depending on the cell type. Typically, CHO cellswill be grown in a medium comprising about 50, 75, 100, 125, 150, 200,300, 500, 1000, 2000, 3000, 4000, 5000 or 6000 nM of MTX for geneamplification.

The fluorescence of the cells may be detected using anyfluorescently-labelled folate analogue that covalently binds to DHFR.Such fluorescent compounds include, e.g., fluorescent methotrexate(f-MTX) or fluorescent trimethoprim (f-TMP).

In step (c), the fluorescence may be measured using any apparatuswell-known in the art such as, e.g., a fluorescence microscope or afluorescence-activated cell sorter (FACS) or the like. Using a FACS isparticularly advantageous when performing high-throughput screenings.

In a preferred embodiment, the 20% of cells that exhibit highestfluorescence in step (c) comprise the cell that exhibits highestexpression of said protein of interest. Preferably, the 10% of cellsthat exhibit highest fluorescence in step (c) comprise the cell thatexhibits highest expression of said protein of interest. Mostpreferably, the 1% or the 5% of cells that exhibit highest fluorescencein step (c) comprise the cell that exhibits highest expression of saidprotein of interest.

Any number of cells may be screened by such a method. Preferably, thefluorescence of at least 1, 20, 50, 100, 500, 1,000, 5,000, 10,000,50,000, 100,000, 500,000, 1,000,000 or 10,000,000 cells is assayed instep (c). Most preferably, a population of cells sufficient forobtaining at least 1,000 to 10,000,000 independent transfectants beingresistant to puromycin is screened. Out of these, at least 10 to1,000,000 candidate clones being resistant to puromycin can further beassayed for fluorescence.

The cells obtained at the end of the above screening method may beranked relative to each other regarding res-DHFR expression. The cellsexhibiting the highest fluorescence may be selected at the end of any ofthe above methods of screening. For example, individual cells exhibitingDHFR activity corresponding to the top 5-20% of res-DHFR expressors areselected for further analysis of expression of the gene of interest in asubsequent step.

In a preferred embodiment, the above screening method further comprisesthe step of (d) selecting about 1% to about 20% of the cells assayed instep (c), wherein the selected cells are those exhibiting highestfluorescence in step (c). About 5% to about 20% of the cells assayed instep (c) may be selected based on highest res-DHFR activity.Alternatively, about 1%, 1.5%, 2%, 3%, 4%, 5% to about 30%, 40%, 50%,60%, 70% or 80% of the cells assayed in step (c) may be selected basedon highest res-DHFR activity.

Steps (b) (i.e. selecting resistant cells), (c) (i.e. assaying thefluorescence) and (d) (i.e. selecting the most fluorescent cells) may beiteratively repeated on the population selected at the end of step (d).For example, at least 2, 3, 5 or 10 iterations may be carried out. Thismay be done with or without changing conditions in between the selectionsteps. Changing conditions may include e.g. increasing MTX concentrationto induce gene amplification or varying culture conditions such as mediacomponents or physico-chemical parameters.

Upon selection of the cells exhibiting the highest fluorescence, theexpression level of the protein of interest in said selected cells mayfurther be assayed.

Then, the about 1% to about 20% of the cells exhibiting the highestexpression of said protein of interest may be selected. For example,about 1%, 1,5%, 2%, 3%, 4%, 5% to about 15%, 18% or 20% of the cellsexhibiting the highest expression of said protein of interest may beselected. Preferably, the cell exhibiting the highest expression of saidprotein of interest is selected. This selection based on expression ofthe protein of interest is preferably performed after the last iterationof step (d) (i.e. the last selection based on fluorescence).

A further aspect of the invention pertains to a method of obtaining acell line expressing a protein of interest, said method comprising thesteps of:

-   -   a) screening cells according to the above method; and    -   b) establishing a cell line from said cells.

As used herein, a “cell line” refers to one specific type of cell thatcan grow in a laboratory. A cell line can usually be grown in apermanently established cell culture, and will proliferate indefinitelygiven appropriate fresh medium and space. Methods of establishing celllines from isolated cells are well-known by those of skill in the art.

Another aspect relates to a method of producing a protein of interest,said method comprising the step of:

-   -   a) culturing a cell line obtained as described above under        conditions which permit expression of said protein of interest;        and    -   b) collecting said protein of interest.

Conditions which permit expression of the protein of interest can easilybe established by one of skill in the art by standard methods. Forexample, the conditions disclosed in Example 3.3.1 may be used.

In a preferred embodiment, the above method of producing a protein ofinterest further comprises the step of purifying said protein ofinterest. The purification may be made by any technique well-known bythose of skill in the art. In the case of a protein of interest for usein the field of pharmaceutics, the protein of interest is preferablyformulated into a pharmaceutical composition.

The invention further pertains to the use of a res-DHFR polypeptide forscreening cells for expression of a protein of interest, to the use of ares-DHFR nucleic acid for screening cells for expression of a protein ofinterest, and to the use of a res-DHFR vector for screening cells forexpression of a protein of interest. The res-DHFR polypeptide, nucleicacid or vector preferably is a Puro-DHFR polypeptide, nucleic acid orvector.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent applications, issuedU.S. or foreign patents or any other references, are entirelyincorporated by reference herein, including all data, tables, figuresand text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplication such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

EXAMPLES Example 1 Protocols

1.1. Construction of the Puro-DHFR Nucleic Acid

The constructs described hereinbelow are depicted in FIG. 1. Allconstructs are based on the pGL3-basic plasmid backbone (Promega).

The fusion protein between the puromycin resistance gene and wild typemurine DHFR was obtained by recombinant PCR. Part of the poliovirus IRESand the complete puromycin resistance gene ORF (omitting the stop codon)that are present in vector pmCMV(IE1)-SEAP-IRES-PuroR-p279 wereamplified by PCR using primers of SEQ ID Nos. 7 and 8 and a highfidelity DNA polymerase (HS-KOD; Novagen). The wild-type murine DHFR ORFand part of the SV40 late polyadenylation signal present in plasmidpSV40-DHFR (p1474) were amplified using primers of SEQ ID Nos. 9 and 10.

To generate the fusion Puro-DHFR marker, the resulting PCR products weremixed and reamplified using primers of SEQ ID Nos. 7 and 10 and clonedinto a vector wherein the murine IE1 promoter drives the expression ofthe human placental alkaline phosphatase gene (SEAP) and the Puro-DHFRselection marker is expressed as the second cistron placed downstream ofthe poliovirus IRES. The integrity of all elements amplified by PCR wasverified by sequencing. This vector is further referred to aspmCMV(IE1)-SEAP-IRES-Puro/DHFR-p325 or p325.

1.2. Transfection and Cell Culture

The protocol for selecting stable transfectants is schematized in FIG.2.

CHO-S cells were derived from the Chinese hamster ovaries and adapted toserum-free suspension culture (Invitrogen/Gibco, La Jolla, Calif.). CHODX11-F10 is a cell line derived from the DHFR-deficient CHO DUKXB11 cellline (Urlaub et al. 1980) that was adapted to growth in suspension inserum-free media. Both cells are routinely cultivated in ProCho5 (LonzaBiologics). The medium for cultivation of DXB11-F10 cells wassupplemented with hypoxanthine (100 μM) and thymidine (16 μM) (HTsupplement; Invitrogen/Gibco) unless the contrary is indicated.

CHO-S and DXB11-F10 cells were transfected using polyethylenimine(linear PEI 25 kd). Cells were plated in 6-well plates in 2.5 mLRPMI-1640 (Invitrogen/Gibco) plus 0.05% Pluronic F68 (Sigma) at aconcentration of 5×10⁵ cells per mL. 5 μg of linearized plasmid DNA in250 μl of 150 mM NaCl was mixed with a solution of 15 μl of 1 mM linearPEI25 diluted in 250 μl of 150 mM NaCl. The PEI:DNA complexes wereallowed to form for 5 minutes at room temperature and are then added tothe cells. After 3 hours the transfection medium is replaced withserum-free culture medium.

48 hours post-transfection selection was applied and the medium changed2 times per week until cells recovered and cell viability was greaterthan 90%.

For selection, puromycin was used at 10 μg/ml and the folate analoguemethotrexate (Calbiochem) was used at a concentration of 50 to 100 nM.

For amplification studies, clones were first obtained by limiteddilution at 0.3 cell per well in 384-well plates under selection withpuromycin at 10 μg/ml. Clones were then cultivated for 4 weeks underselection with puromycin (10 μg/ml) or puromycin plus methotrexate (100nM). Genomic DNA was then extracted and reporter gene copy number wasdetermined by QPCR.

1.3. Determination of Reporter Gene Expression by SEAP Assay

Stably transfected cell pools were seeded at 2.5×10⁵ cells/ml in 125 mlshake-flasks and grown for up to 7 days in batch culture. Cell culturemedia was harvested at various time points and to avoid day-to-dayvariation in the SEAP measurements the samples were kept at −20° C.until analysis. Relative SEAP activity was determined in a kineticenzyme assay. 10 μl of serial dilutions of media in hepes-bufferedsaline solution (HBSS) were added to a 96-well plate then 100 μl of aPhosphatase Substrate Solution (Pierce) was added to each well andreadings of OD at 405 nM were taken at regular time intervals. Only thelinear window of the plot OD vs. time was considered for analysis.

1.4. Determination of Gene Copy Number by QPCR.

Genomic DNA was isolated using the GenElute Mammalian Genomic DNAMiniprep kit (Sigma) according to the manufacturer's instructions andquantified spectrophotometrically. For determination of gene copy number10ng of genomic DNA were analyzed by quantitative PCR with the 7500Real-Time PCR instrument (Applied Biosystems) using standard cyclingconditions in a multiplex assay. A puromycin-specific TaqMan probe wasused to detect the reporter construct and second TaqMan probe, detectingthe hamster glyceraldehyde phosphate dehydrogenase (GAPDH) gene was usedas an endogenous control. A standard curve was generated using genomicDNA from cell lines in which the puromycin gene copy number had beendetermined by Southern blot.

The oligonucleotides had the sequences of SEQ ID Nos. 11, 12 and ofFAM-SEQ ID NO: 13-BHQ1 for detection of the puromycin acetyltransferasegene, and of SEQ ID Nos. 14, 15 and of YY-SEQ ID NO: 16-BHQ1 fordetection of the GAPDH gene. FAM and YY are abbreviations forfluorophores 5-carboxyfluorescein and Yakima Yellow respectively (EpochBiosciences). BHQ1™ (Biosearch Technologies, Inc.) is a quencher linkedto the 3′ of the TaqMan probes.

Core PCR reagents were from Applied Biosystems and 96-well detectionplates were obtained from AxonLab.

1.5. Determination of Relative Reporter Gene Expression by ReverseTranscriptase-QPCR.

Total RNA was isolated from ˜5×10⁶ cells using the NucleoSpin RNA II kit(Macherey-Nagel)-which includes a DNase treatment step—and RNAconcentration was determined spectrophotometrically at 260 nm.

Relative quantification of the reporter expression was performed byOne-Step Reverse Transcriptase-QPCR (One-Step RT-PCR Master Mix Reagent;Applied Biosystems) on 25 ng of total RNA using the Puro and GAPDHprimers and TaqMan probes described above. GAPDH served as an endogenouscontrol. The amounts of the reporter mRNA were calculated by the ΔΔCtmethod and expressed relative to the pool p279 (selected with puromycinonly).

1.6. Labeling with Fluorescein-Methotrexate

2.5-5×10⁵ cells were incubated over-night at 29° C. in 0.5 mL of culturemedium containing 10 mM fluorescein-labeled methotrexate (F-MTX,Molecular Probes/Invitrogen). Labeled cells were washed in serum-freeculture medium and images were recorded using a fluorescence microscope(Olympus CKX41 microscope equipped with a DP50 digital camera) using aFITC filter set.

Example 2 Puro-DHFR is a Quadrifunctional Marker

2.1. Puro-DHFR Confers Resistance to Puromycin to the Transfected Cells(i.e. Puro-DHFR has Puromycin Acetyltransferase Activity)

CHO DXB11-F10 cells were transfected with the p325 vector encodingPuro-DHFR as described in Example 1.2. As shown on FIG. 3, the Puro-DHFRselection marker confers puromycin resistance to the transfected cells.

2.2. Puro-DHFR Allows Growth in Absence of HT (i.e. Puro-DHFR has DHFRActivity)

DHFR-deficient cells are sensitive to MTX and require the presence of HT(Hypoxanthine and Thymidine) in the culture medium for growth.

DHFR-deficient CHO DXB11-F10 cells were transfected with the p325 vectorencoding Puro-DHFR as described in Example 1.2. As shown on FIG. 3, thePuro-DHFR selection marker allows growth of p325-transfected DXB11-F10cells in the absence of HT.

2.3. Puro-DHFR Induces Gene Amplification

CHO-S cells, which endogenously express DHFR, were transfected with thep325 vector encoding Puro-DHFR as described in Example 1.2. The cellswere selected in the presence of puromycin (10 μg/ml). Clones wereobtained by limited dilution of the resistant population. Interestingly,the experiments shown here demonstrate that Puro-DHFR selection andamplification is feasible in CHO-S cells despite its endogenous DHFRbackground expression.

Twenty randomly selected clones were cultivated either in the presenceof puromycin (10 μg/ml) or in the presence of puromycin and 100 nM MTXfor 4 weeks in order to test for gene amplification.

The gene copy number was then determined for pools of transformed cellsas described in Example 1.4. As shown on FIG. 8, amplification of genecopy number depends upon selection with MTX. In 3 of 20 clonesre-selected with puromycin plus MTX at 100 nM (circled) reporter copynumber is increased compared to selection with puromycin only,demonstrating the potential for gene amplification.

2.4. Puro-DHFR can be Detected Through its Fluorescence

FIG. 5 shows labeling with fluorescent methotrexate of CHO DXB11-F10cells transfected with the p325 vector encoding Puro-DHFR. Florescencewas detected as described in Example 1.6. The untransfected DXB11-F10cells are much less fluorescent than the transfected ones. In addition,higher selection pressure in presence of MTX (50 nM) leads to increasedexpression of the puro-DHFR selection marker and more intense celllabeling.

FIG. 7 shows labeling with fluorescent methotrexate of CHO-S cellstransfected with the p325 vector encoding Puro-DHFR. The backgroundlevels of fluorescence in pools of CHO-S p279 (expressing only theendogenous DHFR gene) is significantly lower than fluorescence in poolsof CHO-S p325 cells selected at high levels of MTX.

Thus Puro-DHFR can be detected both in DHFR+ (CHO-S) and in DHFR−(CHO-DXB11-F10) cells.

Example 3 Puro-DHFR is a Surrogate Marker Useful for Screening for CellsExpressing High Levels of a Protein of Interest

3.1. Puro-DHFR Allows Isolating Clones Expressing High Levels of aProtein of Interest

DXB11-F10 cells were transfected with the p325 vector encodingPuro-DHFR. This vector additionally comprises SEAP as a reporter gene(protein of interest). Stable pools were selected in presence or in theabsence of MTX, and expression of SEAP was measured as described inExample 1.3. As shown on FIG. 4, pools selected in the presence of MTXexhibited about 2-fold higher SEAP expression than pools selected withpuromycin or in absence of HT only.

CHO-S cells were transfected either with the p325 vector encodingPuro-DHFR or with the p279 vector encoding puromycin N-acetyltransferaseand SEAP. Stable pools were selected in presence of puromycin and of 0,50 or 100 nM MTX. Expression of SEAP was measured at day 7 both asdescribed in Example 1.3a and in Example 1.5. FIG. 6 shows thatincreased selection pressure of CHO-S p325 stable pool with puromycinplus MTX (100 nM) leads to significantly higher expression of SEAPprotein, both at the protein level (A) and at the mRNA level (B).

Thus the puro-DHFR marker allows isolating clones expressing higherlevels of the SEAP protein than prior art markers such as puromycinN-acetyltransferase.

3.2. Puro-DHFR can be Used as a Surrogate Marker in High-ThroughputScreenings

Puro-DHFR is used as a selective and surrogate marker to establish andscreen candidate clones with a vector expressing both Puro-DHFR and theprotein of interest. After transfection, selection and amplification, aprimary screen is done by FACS for fluorescence (i.e. high Puro-DHFRexpression) with a high probability of selecting clones that alsoexhibit high gene of interest expression. Then a second screen isperformed for expression of the protein of interest, possibly directlyby ELISA.

Using Puro-DHFR in high throughput screening (HTS) thus allows keepingthe same chance for selecting high expressing clones, and allow reducingtime and resources. In addition, it is important to note that the fusionof two individual enzymes with so different activities and originssurprisingly retains their function in Puro-DHFR as it is describedhere. Indeed, Puro-DHFR can truly be used to provide selectivity instable transfection, act as an amplifiable gene, and act as a surrogatemarker for screening candidate clones for high expression of a gene ofinterest.

REFERENCES

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We claim:
 1. An isolated Puro-DHFR polypeptide comprising SEQ ID NO: 2, said polypeptide having: (i) puromycin N-acetyl transferase activity; and (ii) dihydrofolate reductase activity.
 2. A method of producing a protein of interest comprising expressing a first nucleic acid encoding a polypeptide of interest and a second nucleic acid encoding a polypeptide according to claim 1 in a host cell under conditions permitting the expression of said polypeptide of interest.
 3. The method according to claim 2, said method further comprising the purification of said protein of interest.
 4. A method of expressing a polypeptide having puromycin N-acetyl transferase activity and dihydrofolate reductase activity comprising expressing a nucleic acid encoding a polypeptide according to claim 1 in a host cell under conditions permitting the expression of said polypeptide. 